Bottom Line:
Investigations involving pyrazoleamide-resistant parasites, whole-genome sequencing and gene transfers reveal that mutations in two proteins, a calcium-dependent protein kinase (PfCDPK5) and a P-type cation-ATPase (PfATP4), are necessary to impart full resistance to these compounds.A pyrazoleamide compound causes a rapid disruption of Na(+) regulation in blood-stage Plasmodium falciparum parasites.Similar effect on Na(+) homeostasis was recently reported for spiroindolones, which are antimalarials of a chemical class quite distinct from pyrazoleamides.

ABSTRACTThe quest for new antimalarial drugs, especially those with novel modes of action, is essential in the face of emerging drug-resistant parasites. Here we describe a new chemical class of molecules, pyrazoleamides, with potent activity against human malaria parasites and showing remarkably rapid parasite clearance in an in vivo model. Investigations involving pyrazoleamide-resistant parasites, whole-genome sequencing and gene transfers reveal that mutations in two proteins, a calcium-dependent protein kinase (PfCDPK5) and a P-type cation-ATPase (PfATP4), are necessary to impart full resistance to these compounds. A pyrazoleamide compound causes a rapid disruption of Na(+) regulation in blood-stage Plasmodium falciparum parasites. Similar effect on Na(+) homeostasis was recently reported for spiroindolones, which are antimalarials of a chemical class quite distinct from pyrazoleamides. Our results reveal that disruption of Na(+) homeostasis in malaria parasites is a promising mode of antimalarial action mediated by at least two distinct chemical classes.

f1: Structure and antimalarial activities of pyrazoleamide compounds.(a) Structures of the hit and lead compounds. (b) Growth inhibition assays of PA21A092 against the indicated stages of Dd2 line of P. falciparum. (c) PA21A092 inhibits male (blue) and female (red) gamete production by mature gametocytes. EC50 values are estimated to be 39 and 74 nM for male and female gamete production, respectively; methylene blue as a positive control had EC50 values of 39 and 43 nM, respectively, in these assays. (d) PA21A092 is active against clinical field isolates of P. falciparum (red circles) and P. vivax (blue circles) in ex vivo growth inhibition assays with equal potency against ring (closed triangles) and trophozoite (open triangles) stages in both species.

Mentions:
The initial hit compounds C416, a pyrazoleurea derivative, and C2-1, a pyrazoleamide derivative, (Fig. 1a) were identified through a structure-based in silico screening of a compound library11 and showed growth inhibitory activity with effective concentration for 50% growth inhibition (EC50) of 150 and 50 nM, respectively, against P. falciparum. An extensive medicinal chemistry campaign (to be described in detail elsewhere) was conducted by synthesizing variants of pyrazoleamide compound C2-1 and leading to a series of compounds with low nanomolar activity against P. falciparum. Structures of three of the late lead compounds, PA21A050 (EC50: 0.7 nM), PA21A092 (EC50: 5 nM) and PA21A102 (EC50: 8 nM), are shown in Fig. 1a. On the basis of its biological, pharmaceutical and toxicological profiles, compound PA21A092 was designated as a preclinical drug candidate to be developed for first-in-human studies. Relatively equal activity of PA21A092 was observed over a 48-h period against P. falciparum, regardless of the stage of parasites used in the assay, with EC50 values ranging from 5 to 13 nM (Fig. 1b). The functional viability of P. falciparum mature Stage V male and female gametocytes as manifested by their ability to form male and female gametes, respectively, was inhibited when exposed to PA21A092 with an EC50 of 39 and 74 nM, respectively (Fig. 1c), indicating its potential to act also as a transmission-blocking drug. A panel of eight P. falciparum lines resistant to a number of currently used antimalarial drugs were susceptible to compound 21A092 (Supplementary Table 1), suggesting a mode of action different from currently used antimalarials. Furthermore, PA21A092 was tested against clinical isolates of P. falciparum and P. vivax infecting patients living in an area with high prevalence of multiple drug resistance. Using an ex vivo assay12, we found that both species were highly susceptible to PA21A092, with a median EC50 of 18 nM against P. falciparum from 32 patients and 10 nM against P. vivax from 35 patients (Fig. 1d).

f1: Structure and antimalarial activities of pyrazoleamide compounds.(a) Structures of the hit and lead compounds. (b) Growth inhibition assays of PA21A092 against the indicated stages of Dd2 line of P. falciparum. (c) PA21A092 inhibits male (blue) and female (red) gamete production by mature gametocytes. EC50 values are estimated to be 39 and 74 nM for male and female gamete production, respectively; methylene blue as a positive control had EC50 values of 39 and 43 nM, respectively, in these assays. (d) PA21A092 is active against clinical field isolates of P. falciparum (red circles) and P. vivax (blue circles) in ex vivo growth inhibition assays with equal potency against ring (closed triangles) and trophozoite (open triangles) stages in both species.

Mentions:
The initial hit compounds C416, a pyrazoleurea derivative, and C2-1, a pyrazoleamide derivative, (Fig. 1a) were identified through a structure-based in silico screening of a compound library11 and showed growth inhibitory activity with effective concentration for 50% growth inhibition (EC50) of 150 and 50 nM, respectively, against P. falciparum. An extensive medicinal chemistry campaign (to be described in detail elsewhere) was conducted by synthesizing variants of pyrazoleamide compound C2-1 and leading to a series of compounds with low nanomolar activity against P. falciparum. Structures of three of the late lead compounds, PA21A050 (EC50: 0.7 nM), PA21A092 (EC50: 5 nM) and PA21A102 (EC50: 8 nM), are shown in Fig. 1a. On the basis of its biological, pharmaceutical and toxicological profiles, compound PA21A092 was designated as a preclinical drug candidate to be developed for first-in-human studies. Relatively equal activity of PA21A092 was observed over a 48-h period against P. falciparum, regardless of the stage of parasites used in the assay, with EC50 values ranging from 5 to 13 nM (Fig. 1b). The functional viability of P. falciparum mature Stage V male and female gametocytes as manifested by their ability to form male and female gametes, respectively, was inhibited when exposed to PA21A092 with an EC50 of 39 and 74 nM, respectively (Fig. 1c), indicating its potential to act also as a transmission-blocking drug. A panel of eight P. falciparum lines resistant to a number of currently used antimalarial drugs were susceptible to compound 21A092 (Supplementary Table 1), suggesting a mode of action different from currently used antimalarials. Furthermore, PA21A092 was tested against clinical isolates of P. falciparum and P. vivax infecting patients living in an area with high prevalence of multiple drug resistance. Using an ex vivo assay12, we found that both species were highly susceptible to PA21A092, with a median EC50 of 18 nM against P. falciparum from 32 patients and 10 nM against P. vivax from 35 patients (Fig. 1d).

Bottom Line:
Investigations involving pyrazoleamide-resistant parasites, whole-genome sequencing and gene transfers reveal that mutations in two proteins, a calcium-dependent protein kinase (PfCDPK5) and a P-type cation-ATPase (PfATP4), are necessary to impart full resistance to these compounds.A pyrazoleamide compound causes a rapid disruption of Na(+) regulation in blood-stage Plasmodium falciparum parasites.Similar effect on Na(+) homeostasis was recently reported for spiroindolones, which are antimalarials of a chemical class quite distinct from pyrazoleamides.

ABSTRACTThe quest for new antimalarial drugs, especially those with novel modes of action, is essential in the face of emerging drug-resistant parasites. Here we describe a new chemical class of molecules, pyrazoleamides, with potent activity against human malaria parasites and showing remarkably rapid parasite clearance in an in vivo model. Investigations involving pyrazoleamide-resistant parasites, whole-genome sequencing and gene transfers reveal that mutations in two proteins, a calcium-dependent protein kinase (PfCDPK5) and a P-type cation-ATPase (PfATP4), are necessary to impart full resistance to these compounds. A pyrazoleamide compound causes a rapid disruption of Na(+) regulation in blood-stage Plasmodium falciparum parasites. Similar effect on Na(+) homeostasis was recently reported for spiroindolones, which are antimalarials of a chemical class quite distinct from pyrazoleamides. Our results reveal that disruption of Na(+) homeostasis in malaria parasites is a promising mode of antimalarial action mediated by at least two distinct chemical classes.